Multiple Sclerosis
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) and is second only to trauma as the major cause of acquired disability in young adult Caucasian populations. The disease usually starts between 20 to 40 years of age and there are two major forms. Relapsing-remitting MS (RR-MS) is the most frequent form (85%-90%) and affects women about twice as often as men. Most RR-MS patients later develop the second major form known as secondary progressive MS (SP-MS). About 10%-15% of patients show a steady progression following disease onset with the absence of relapses, termed primary progressive PP-MS. (Sospedra, M. and Martin, R. Immunology of multiple sclerosis. Annu Rev Immunol 23, 683 (2005)). MS is a highly heterogeneous disease where every patient differs in clinical presentation and response to treatments.
Disease activity in MS can be defined by a number of different parameters such as changes in number of gadolinium enhancing lesions (Katz, D. et al., Correlation between magnetic resonance imaging findings and lesion development in chronic, active multiple sclerosis. Ann Neurol 34 (5), 661 (1993)), Expanded Disability Status Scale (EDSS) scores and relapse rate. Gadolinium enhancing lesions imaged by Magnetic Resonance Imaging (MRI) is one of the most reliable indications of active inflammation in MS (see, Katz, Id.). However the MRI only monitors structural damage occurring in the brain, while much of the disease activity may be occurring in the spinal cord. In addition, while the MRI gives a clear indication of damage occurring in the brain, a biomarker has the advantage in that it can provide quantitative and more accurate measurements of disease activity. The discovery of a biomarker such as Fetuin-A protein which can consistently be used to predict the level of disease activity could translate to faster and more accurate therapeutic decisions by physicians and healthcare providers.
Fetuin-A
Fetuin-A, also known as Alpha2 HS-glycoprotein, is a major serum protein in mammals mainly of hepatic origin being 95% liver-derived (Triffitt, J. T. et al., Origin of plasma alpha2HS-glycoprotein and its accumulation in bone. Nature 262 (5565), 226 (1976)). Expression in other cell types such as cells of monocyte/macrophage lineage during development and in the adult bone marrow has also been described. (Dziegielewska, K. et al., The expression of fetuin in the development and maturation of the hemopoietic and immune systems. Histochem Cell Biol 106 (3), 319 (1996)). Fetuin-A is a member of a family of related glycoproteins that belong to the cystatin superfamily (Kellermann, J., Haupt. H., Auerswald, E. A., and Muller-Ester, W., The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens. J Biol Chem 264 (24), 14121 (1989). It has a diverse range of biological functions including osteogenesis and bone resorption (Szweras, M. et al., alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling. J Biol Chem 277 (22), 19991 (2002)), regulation of insulin activity (Mathews, S. T. et al., Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes 51 (8), 2450 (2002)), and inhibition of unwanted mineralization (Heiss, A. et al., Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J Biol Chem 278 (15), 13333 (2003); Schafer, C. et al., The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest 112 (3), 357 (2003); Schinke, T. et al., The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis. J Biol Chem 271 (34), 20789 (1996)).
Fetuin-A knockout mice are fertile and show no gross anatomical abnormalities. There is however compromised serum inhibition of apatite formation and some animals develop ectopic microcalcifications in soft tissues, corroborating a role for fetuin in serum calcium homeostasis (Jahnen-Dechent, W. et al., Cloning and targeted deletion of the mouse fetuin gene. J Biol Chem 272 (50), 31496 (1997)). Annexin II and VI are the putative cell surface receptors for fetuin-A and require the presence of calcium ions for binding (Kundranda, M. N. et al., Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A. Biochim Biophys Acta 1693 (2), 111 (2004)). Integrins have been implicated as possible fetuin-A receptors since addition of antibody against β1 integrin substantially reduced the adherence of tumor cells to immobilized Fetuin-A in the presence of magnesium ions. (see, Kundranda, Id.)
Of particular interest is the immune regulatory functions of fetuin-A. It is classified as a negative acute-phase protein since its concentration in serum is down-regulated during episodes of trauma and acute inflammation (Lebreton, J. P. et al. Serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant. J Clin Invest 64 (4), 1118 (1979)). Fetuin has anti-inflammatory properties in that it attenuates TNF-α synthesis by LPS-stimulated macrophages (Dziegielewska, K. M., Andersen, N. A., and Saunders, N. R., Modification of macrophage response to lipopolysaccharide by fetuin. Immunol Lett 60 (1), 31 (1998)) and in an LPS-independent model of acute inflammation (Ombrellino, M. et al., Fetuin, a negative acute phase protein, attenuates TNF synthesis and the innate inflammatory response to carrageenan. Shock 15 (3), 181 (2001)).
Fetuin-A is also a TGF-β antagonist, and binds directly to TGF-β1 and TGF-β2. Fetuin-A blocks binding of TGF-β1 to the TGF-β receptor type 2 and therefore inhibits signaling through this major receptor. (Demetriou, M. et al., Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist. J Biol Chem 271 (22), 12755 (1996)). Interestingly, a prior study showed that lower levels of TGF-β are present in the cerebrospinal fluid (“CSF”) of MS patients during periods of disease activity when compared to periods of remission or inactivity (Carrieri, P. B. et al., Possible role of transforming growth factor-beta in relapsing-remitting multiple sclerosis. Neurol Res 19 (6), 599 (1997)). Since fetuin-A is an antagonist of TGF-β it is plausible that high levels of fetuin-A may affect levels and/or the activity of TGF-β. Reduced TGF-β activity could contribute to central nervous system (“CNS”) inflammation since it is a potent immunosuppressor (Moustakas, A., Pardali, K., Gaal, A., and Heldin, C. H., Mechanisms of TGF-beta signaling in regulation of cell growth and differentiation. Immunol Lett 82 (1-2), 85 (2002) and suppresses the growth and differentiation of most immune cell lineages including B and T cells (Letterio, J. J. and Roberts, A. B., Regulation of immune responses by TGF-beta. Annu Rev Immunol 16, 137 (1998)).
MS pathology is characterized by blood brain barrier (“BBB”) breakdown which leads to the infiltration of macrophages and lymphocytes into the CNS resulting in areas of demyelination or plaques. Matrix metalloproteinases (“MMPs”) are a family of enzymes that degrade specific components of the extracellular matrix and have been implicated in the breakdown of and transmigration of immune cells across the BBB in MS (Leppert, D. et al., T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol 154 (9), 4379 (1995); Stuve, O. et al., Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9. Ann Neurol 40 (6), 853 (1996); Lukes, A., Mun-Bryce, S., Lukes, M., and Rosenberg, G. A., Extracellular matrix degradation by metalloproteinases and central nervous system diseases. Mol Neurobiol 19 (3), 267 (1999)). Fetuin-A has been shown to associate with MMPs with the strongest association being with MMP-9 (Ochieng. J. and Green, B. The interactions of alpha 2HS glycoprotein with metalloproteinases. Biochem Mol Biol Int 40 (1), 13 (1996)). MMP-9 is produced by cells of the monocyte lineage and exists in an inactive form (proMMP-9) which is cleaved by proteinases to produce the active form. Fetuin-A was found to stimulate the release of pro-MMP-9 from a human monocytic cell line and from freshly isolated human peripheral blood monocytes, as well as activate proMMP-9 present in the THP-1 conditioned media (Tajirian, T., Dennis, J. W., and Swallow, C. J., Regulation of human monocyte proMMP-9 production by fetuin, an endogenous TGF-beta antagonist. J Cell Physiol 185 (2), 174 (2000)). It was shown that TGF-β1 has an inhibitory effect on the release of proMMP-9, and therefore fetuin-A being an antagonist of TGF-β1 can oppose this inhibitory effect on proMMP-9 release. This data suggests that under physiological conditions fetuin-A can contribute to matrix degradation.
Biomarkers
Biomarkers can be defined as biological molecules that are indicators of physiologic state and also of change during a disease process (Srinivas, P. R., Kramer, B. S., and Srivastava. S., Trends in biomarker research for cancer detection. Lancet Oncol 2 (11), 698 (2001)). A biomarker is only useful if it can be used to provide an early indication of the disease, if it can monitor disease progression, if it can be easily detected and if it can be a factor measurable across populations. The discovery of a reliable biomarker in MS is still an area of active research but the identification of reliable biomarkers can provide insight into the underlying mechanisms of disease progression and help to better predict the disease course. Over the years several groups have reported different proteins that they believed to be important biomarkers including the chemokines CXCL10, CCL2 (Sorensen, T. L. et al., Chemokines CXCL10 and CCL2: differential involvement in intrathecal inflammation in multiple sclerosis. Eur J Neurol 8 (6), 665 (2001)), and MMPs and tissue-inhibitors to metalloproteinases (TIMPs) (Rosenberg, G. A., Matrix metalloproteinases biomarkers in multiple sclerosis. Lancet 365 (9467), 1291 (2005)). One of the major challenges in biomarker discovery is obtaining an ample sample size and having uniformity in classification of patients. An ideal biomarker can be used for classification of MS patients, selecting the optimal course of treatment and for monitoring the response to those treatments.